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Basic fibroblast growth factor enhances cell proliferation in the dentate gyrus of neonatal rats following hypoxic–ischemic brain damage
Neuroscience Letters 673 (2018) 67–72
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Basic fibroblast growth factor enhances cell proliferation in the dentate gyrus of neonatal rats following hypoxic–ischemic brain damage ⁎
Huan Zhua,1, Lixing Qiaoa,1, Yao Sunb, Liping Yina, Li Huanga, Li Jianga, , Jiaqing Lic,
T
⁎
a
Department of Pediatrics, Zhongda Hospital, Southeast University, Nanjing, Jiangsu Province 210009, China Rehabilitation Department, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu Province 210008, China c Department of Anesthesiology, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu Province 210008, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cell proliferation Dentate gyrus Neonatal HIBD bFGF Cerebral hypoxia Cerebral ischemia
Background: Perinatal hypoxic–ischemic insult is considered a major contributor to child mortality and morbidity and leads to neurological deficits in newborn infants. There has been a lack of promising neurotherapeutic interventions for hypoxic–ischemic brain damage (HIBD) for clinical application in infants. The present study aimed to investigate the correlation between neurogenesis and basic fibroblast growth factor (bFGF) in the hippocampal dentate gyrus (DG) region in neonatal rats following HIBD. Material and methods: Cell proliferation was examined by detecting BrdU signals, and the role of bFGF in cell proliferation in the DG region following neonatal HIBD was investigated. Results: Cell proliferation was induced by HIBD in the hippocampal DG of neonatal rats. Furthermore, bFGF gene expression was upregulated in the hippocampus in neonatal rats, particularly between 7 and 14 days after HIBD. Moreover, intraperitoneal injection of exogenous bFGF enhanced cell proliferation in the hippocampal DG following neonatal HIBD. Conclusions: Taken together, these data indicate that cell proliferation in the DG could be induced by neonatal HIBD, and bFGF promotes proliferation following neonatal HIBD.
1. Introduction Perinatal hypoxic–ischemic injury leads to neurological deficits in newborn infants and is considered a major contributor to child mortality and morbidity [20]. Hypoxic–ischemic brain damage (HIBD) encompasses complex pathological and cellular injuries to the brain caused by hypoxia, ischemia, cytotoxicity, or a combination of these conditions [21]. Despite significant progress in elucidating the mechanism(s) underlying hypoxia–ischemia (H-I), there is currently a lack of promising neurotherapeutic interventions following HIBD for clinical application in infants. It is well-known that the brain has some capacity for neurogenesis in response to various types of damage that result in the loss of neurons in adult mammalian brains following H-I, particularly in two regions, the dentate gyrus (DG) subgranular zone (SGZ) and subventricular zone in the lateral ventricles [11]. Endogenous neurogenesis after ischemic injury produces new cells that can integrate into neural networks and play active roles in the recovery from neurological deficits [6]. However, there is contradictory evidence as to whether neonatal H-I increases or decreases the number of hippocampal precursor cells ⁎
1
[3,7,25]. Endogenous neurogenic capacity depends on not only the intrinsic properties of neural precursor cells (NPCs) but also the regional differences in extrinsic regulatory signals and receptor expression. Therefore, harnessing the abilities of such extrinsic factors to amplify neurogenesis represents a neurotherapy for neonatal H-I. The basic fibroblast growth factor (bFGF, also called FGF2) is a member of the FGF superfamily proteins. It binds to the tyrosine kinase receptors FGFR1–4 to activate downstream signaling [24]. bFGF plays an important role in neurogenesis and also promotes neural stem cell (NSC) proliferation and differentiation into neurons during development as well as in the adult mouse brain via pharmacological addition or blockade of bFGF [4,12,22]. However, bFGF-deficient mice exhibit no obvious deficit in NSC proliferation in the adult DG but exhibit impairments in the differentiation of NPCs [23]. bFGF efficiently promotes the regeneration of neurons after injury [2,14] and has also been shown to modulate axonal branching and synaptic plasticity in vitro and in vivo [1,18]. As a neurotrophic factor, bFGF is highly significant for the development of therapies as it has the potential to restore neurological deficits in neurodegenerative diseases and acute stress [8,9,15]. However, the role
Corresponding authors. E-mail addresses: [email protected] (L. Jiang), [email protected] (J. Li). Co-first authors.
https://doi.org/10.1016/j.neulet.2018.01.046 Received 22 September 2017; Received in revised form 18 January 2018; Accepted 23 January 2018 Available online 20 March 2018 0304-3940/ © 2018 Elsevier B.V. All rights reserved.
Neuroscience Letters 673 (2018) 67–72
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chosen for analysis. BrdU-positive cells were counted from the DG region in four 400 × magnification images in each section of the brain at the different time points.
of bFGF in neurogenesis following neonatal H-I remains unexplored. The present study aimed to investigate the correlation between neurogenesis and bFGF in the hippocampal DG region of neonatal rats following H-I. Our data indicated that cell proliferation in the DG is induced by neonatal H-I and that cell proliferation is promoted by bFGF following neonatal H-I. bFGF may have neuroprotective and neurorestorative properties for neurological deficits following neonatal H-I.
2.4. RT-PCR Isolation of total mRNA from the hippocampus was performed using TriPure reagent, following the one-step guanidinium isothiocyanate and acidified phenol method according to the manufacturer’s instructions. RNA concentration was determined using optical density measurements at 260 nm, and RNA purity and integrity were identified using an ultraviolet spectrometer and agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg of total RNA in a 20-μL reaction volume by reverse transcription insolution (4 μL MgCl2, 2 μL dNTP mixture, 1 μL OligodT-Adaptor primer, 0.5 μL RNase inhibitor, and 1 μL reverse transcriptase) at 42 °C for 30 min, 99 °C for 5 min, and 5 °C for 5 min. Ten microliters of product of the reverse transcription reaction were used as a template. PCR was run at 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 60 s, and finally at 72 °C for 8 min. Forward (5′-CTT TGG GTG GAA GGC TGG TCG-3′) and reverse (5′-TGC GGG AAG CGA AGT GAT GC-3′) primers were used to amplify a 448 bp rat bFGF fragment, and 308 bp GADPH served as a control with the following primers: sense 5′- TCA CTC AAG ATT GTC AGC AA-3′ and antisense 5′- AGA TCC ACG ACG GAC ACA TT-3′. Equal amounts of corresponding bFGF and GADPH products were loaded onto 1.5% agarose gels, and the optical densities of ethidium bromide-stained DNA bands were used to quantify the expression of the bFGF gene.
2. Material and methods 2.1. Materials Postnatal day 7 Sprague–Dawley (SD) rats (weighing 12.70 ± 1.14 g; male and female) were purchased from the Animal Laboratory at the Medical College of Southeast University (Nanjing, China). BrdU In-Situ Detection Kits were purchased from BD Bioscience. Oligonucleotide primers for bFGF and GADPH used for RT-PCR were synthesized by SANGON Co., Ltd. TriPure isolation reagent was purchased from Roche Diagnostics Corp. RNA PCR Kits (AMV) version 3.0 were purchased from TaKaRa Biotechnology Co., Ltd (Dalian). 2.2. Animals Procedures for the use of laboratory animals were approved by the institutional animal use and care committee, and the methods were conducted in accordance with the approved guidelines. For immunohistochemistry (IHC), SD rats were randomly divided into six groups (eight rats per group): sham, hypoxia, ischemia, H-I, normal saline (NS), and bFGF groups. Exogenous bFGF (10 μg/kg) was intraperitoneally injected every day for 7 days following H-I in the bFGF group, and saline was used as a control in the NS group. The rats were intraperitoneally injected with 100 mg/kg BrdU twice every week 48 h after different injuries. For RT-PCR, SD rats were randomly divided into four groups: sham, hypoxia, ischemia, and H-I groups. Animals were not injected with BrdU for RT-PCR analysis. H-I was induced according to previously described methods [13]. Seven-day-old SD rats were anesthetized by inhaling diethyl ether. The left common carotid artery was isolated and ligated using 4-0 type silk. The rats were placed in a hypoxic chamber (8% oxygen, 92% nitrogen) for 2 h, then in enclosed, vented chambers that were partially submerged in water (37.0 °C). Animals in the sham group were subjected to isolation but no ligation and no subsequent hypoxia, animals in the hypoxia group were only exposed to hypoxia and not ligation, and animals in the ischemia group were ligated but not exposed to hypoxia.
2.5. Statistical analysis Data are presented as mean ± standard deviation. Statistical comparisons were made using analysis of variance. The Student–Newman–Keuls q-test was used for comparisons between two groups. P < .05 was considered statistically significant. 3. Results 3.1. Pathological changes and apoptosis of brain cells following H-I The H-I model was produced according to the traditional Rice model [13]. Carotid artery ligation caused the ipsilateral eye to remain unopened after 90 days following H-I (Fig. 1a). Brains were collected, and atrophy of the left brain was observed (Fig. 1b). Brain damage of experimental animals in each group was compared at different time points, and hippocampal histopathological changes in each group were detected by the HE staining method. No evident damage was found in the control and sham operated groups (Fig. 1c). However, HE staining revealed edema and apoptosis of brain cells in the hippocampus, and the number of neurons was decreased at 24 h following H-I (Fig. 1d–f). The volume of the hippocampus was significantly reduced, and the pyramidal cells were randomly arranged. Necrosis was also observed after 5 days in the H-I model (Fig. 1g and h). The above data indicate neuronal damage in the neonatal brain following H-I.
2.3. Immunohistochemistry and hematoxylin and eosin (HE) staining The rats were sacrificed at 3, 7, 14, 21 days after injury for IHC and at 0 h, 6 h, 24 h, 48 h, 5 days, 7 days, and 90 days (n = 8) after injury for HE staining. Brains were removed, postfixed in 4% paraformaldehyde, and then cut into sections. Paraffin-embedded tissue was cut to 4μm-thick sections. For HE staining, sections were deparaffinized using xylene, rehydrated, stained using HE, dehydrated with alcohol, and cleared in xylene. Paraffin was removed from the sections using xylene according to the instructions on the BrdU In-Situ Detection Kit. Endogenous peroxidase activity was blocked in the sections. Antigen retrieval was performed using a microwave method. Biotinylated antiBrdU antibodies were applied to the sections on the slide and incubated for 1 h at 37 °C. Ready-to-use streptavidin–HRP was then applied to each slide and incubated for 30 min at room temperature. DAB substrates were stained in hematoxylin. Sections were dehydrated, cleared, and mounted. Results were considered positive when the cell nuclei were stained dark brown. Phosphate-buffered saline was used as a negative control in the procedure. The small intestines of BALB/c mice were stained as a positive control. For cell number quantitation, sections at 80 μm-intervals were
3.2. Time course of cell proliferation in the hippocampus of neonatal brains following H-I Endogenous neurogenesis was induced after ischemic injury in the adult mice brain. In order to investigate whether neurogenesis was also stimulated in neonatal brains following H-I, animals were intraperitoneally injected with BrdU and then sacrificed at specific time points after H-I. The number of newly generated cells was estimated in the entire DG region of the hippocampus by detecting the BrdU signal. The majority of BrdU-positive cells in the DG showed a dark brown color in the nuclei (Fig. 2a–d). There was no pronounced change in the 68
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Fig. 1. Pathological changes and apoptosis of brain cells following hypoxia–ischemia (H-I). (a) Carotid artery ligation caused the ipsilateral eye to remain unopened after 90 days following H-I. (b) Atrophy of the left brain after 90 days following H-I. (c) No evident damage was observed in the hippocampal dentate gyrus in the control and the sham operated groups. (d–f) Hematoxylin and eosin staining reveals edema and apoptosis of brain cells in the hippocampus and a decrease in the number of neurons at 24 h following H-I. Arrows indicate apoptotic bodies. (g and h) Reduction of the hippocampal volume and random pyramidal cell arrangement after 5 days following the H-I model. Necrosis was also observed. Arrows indicate apoptotic bodies. Scale bar represents 10 μm.
markedly reduced 14 days after H-I.
number of BrdU-labeled cells in the hippocampal DG of rats in the sham operated group at different time points (Fig. 2e). The number of BrdUpositive cells was significantly increased in the hippocampal DG of the hypoxia and ischemia groups at 7 or 14 days following damage (182.7% and 146.2% at 7 and 14 days, respectively, after hypoxia vs 3 days, P < .001; 204.5% and 161.6% at 7 and 14 days, respectively, after ischemia vs 3 days, P < .001), and H-I induced a greater number of BrdU-positive cells at different time points (184.9% vs sham group at 3 days, P < .001; 287.2% vs sham group at 7 days, P < .001; 236.4% vs sham group at 14 days, P < .001; 138.6% vs sham group at 21 days, P < .01) (Fig. 2e and Table 1). There was also a significantly higher density of BrdU incorporation between 7 and 14 days following H-I (217% and 195.4% at 7 and 14 days, respectively, after H-I vs at 3 days, P < .001) (Fig. 2 and Table 1). Taken together, these data indicate that cell proliferation was induced by H-I in the hippocampal DG of neonatal rats. Cell proliferation shows increased dynamics after 7 days and then
3.3. Increased expression of bFGF mRNA in the DG following H-I As a potent stem cell mitogen, bFGF promotes neurogenesis during development and in adult mice after injury. Acute stress increases astrocytic bFGF expression to mediate hippocampal cell proliferation [8]. We examined the expression of bFGF mRNA levels in the hippocampus following damage. There was no pronounced change in the bFGF mRNA levels in the hippocampus of rats in the sham operated group at different time points (Fig. 3a and e). bFGF mRNA levels were markedly increased in the hippocampus of the hypoxia and ischemia groups after 7 or 14 days following damage (169.6% and 134.2% at 7 and 14 d, respectively, after hypoxia vs 3d, P < .001 and P < .01; 208.1% and 141.3% at 7 and 14 days, respectively, after ischemia vs 3 days, P < .001 or P < .01), and H-I induced higher bFGF mRNA levels at 69
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Fig. 2. Time course of cell proliferation in the hippocampus of the neonatal brain following hypoxia–ischemia (H-I). (a–d) BrdU-positive cells in the hippocampal dentate gyrus (DG) at 3 days (a), 7 days (b), 14 days (c), and 21 days (d) following H-I. (e) Quantification of the number of BrdU-positive cells in the hippocampal DG at different time points following damage. H-I induced a greater number of BrdU-positive cells at different time points. The highest density of BrdU incorporation was detected between 7 and 14 days following H-I. **P < .01 ***P < .001 vs sham operated group, ## P < .01 ### P < .001 vs 3 days or 21 days, n = 8. Scale bar represents 10 μm. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
Table 1 The number of BrdU-positive cells in the left DG at different time points following damage (x ± S, N = 8). group
The number of BrdU-positive cell 3d
sham hypoxic ischemic hypoxic-ischemic
13.25 13.00 14.00 24.50
7d ± ± ± ±
3.84 3.74 4.50 5.07***
18.50 23.75 28.63 53.13
14 d ± ± ± ±
3.59 4.17***,### 4.89***,### 5.44***,###
20.25 19.00 22.63 47.88
21 d ± ± ± ±
4.40 3.93### 3.54### 5.25***,###
14.25 14.25 12.88 19.75
± ± ± ±
3.81 3.92 4.36 4.53**
** P < .01. *** P < .001 vs sham. ### P < .001 vs 3d or 21d.
neurogenesis following neonatal H-I, rats were intraperitoneally injected with bFGF for 7 days following H-I, and saline was injected in the control group. Brains were removed, and BrdU-labeled cells were detected in the hippocampus. Exogenous bFGF slightly increased the number of BrdU-labeled cells in the hippocampus at 7 and 14 days following H-I compared with those in the H-I and NS groups (119.8% vs H-I group, 121.9% vs NS group at 7 days, P < .05) (Fig. 4). This suggested that exogenous bFGF promotes cell proliferation in the hippocampal DG following neonatal H-I. Taken together, these data indicated that cell proliferation in the DG may be induced by neonatal H-I and that bFGF promotes proliferation following neonatal H-I.
different time points (149.4% vs sham group at 3 days, P < .001; 302.0% vs sham group at 7 days, P < .001; 256.6% vs sham group at 14 days, P < .001; 137.3% vs sham group at 21 days, P < .01) (Fig. 3b–e). Furthermore, the highest levels of bFGF mRNA were detected between 7 and 14 days following H-I (208.9% and 198.9% at 7 and 14 days, respectively, after H-I vs at 3 days, P < .001) (Fig. 3d and e). These data indicated that bFGF gene expression is upregulated in the hippocampus following H-I in neonatal rats, particularly between 7 and 14 days after H-I, which is consistent with the time course of cell proliferation indicated by BrdU incorporation. 3.4. Exogenous bFGF promotes cell proliferation following neonatal H-I In order to investigate whether bFGF also plays a role in 70
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Fig. 3. Increase in the expression of basic fibroblast growth factor (bFGF) mRNA levels in the dentate gyrus following hypoxia–ischemia (H-I). (a) No evident change in bFGF mRNA levels in the hippocampus of rats in the sham operated group at different time points. (b–d) Increase in the expression of bFGF mRNA levels in the hippocampus of hypoxia, ischemia, and H-I groups after 7 or 14 days following damage. (e) Quantification of bFGF mRNA levels at different time points in the hippocampus following damage. H-I induced a higher level of bFGF mRNA at different time points. The highest level of bFGF mRNA was detected between 7 and 14 days following H-I. GAPDH was used as a loading control. **P < .01 ***P < .001 vs sham operated group, ## P < .01### P < .001 vs 3 days or 21 days, n = 4.
4. Discussion
Therefore, it is usually used for birth dating and identification of proliferating cells. BrdU incorporation into DNA produces toxic and mutagenic side effects. The integration of a bromine atom into the DNA alters its stability, increasing the risk of sister-chromatid exchanges, mutations, DNA double-strand breaks and also lengthening the cell cycle of the cells that incorporate it. BrdU staining can be performed by immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BrdU [17]. BrdU immunohistochemistry is the most popular technique to confirm neurogenesis in the adult mammalian brain, including in humans; therefore, we used this technique to provide evidence for an increase in cell proliferation in the neonatal hippocampus following H-I. Most research has focused on the hippocampus to study neurogenesis during development and in the adult brain following injury and further investigate the susceptibility to damage and regenerative capacity due to NSCs in the hippocampus [3,7,11,25]. In the mammalian brain, proliferation is maintained at a high level in the early postnatal brain, particularly in the hippocampus, cerebellum, and olfactory bulb.
H-I is a major cause of morbidity and mortality in the perinatal period, with an incidence of 1/4000 live births [19]. To date, there is sufficient evidence showing the etiology of perinatal asphyxial brain injury, and significant progress has been made to understand the mechanism(s) underlying H-I. However, promising neurotherapeutic interventions after damage must be developed for clinical application in infants. There is increasing evidence that indicates a significant increase in neurogenesis following certain types of damage, such as asphyctic, ischemic, traumatic, and epileptogenic brain injuries, indicating that instead of minimizing cell death, neurological deficits may be improved by stimulation of endogenous neurogenesis after injury [5]. In the present study, we observed that cell proliferation in the DG may be induced after neonatal H-I and that bFGF promoted this cell proliferation. BrdU is a thymidine analog that can incorporate into newly synthesized DNA of dividing cells during the S-phase of the cell cycle.
Fig. 4. Exogenous basic fibroblast growth factor (bFGF) promotes cell proliferation following neonatal hypoxia–ischemia (H-I). Quantification of the number of BrdU-positive cells in the hippocampal dentate gyrus of bFGF-injected mice at different time points following damage. Exogenous bFGF slightly increased the number of BrdU-labeled cells in the hippocampus at 7 d following H-I, compared with those in the H-I and normal saline (NS) groups. **P < .01 ***P < .001 vs sham operated group, ## P < .01 ### P < .001 vs 3 days or 21 days, and *P < .05 vs NS group, N = 8.
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Acknowledgement
The DG produces significant numbers of new neurons throughout life. There is inconsistent evidence regarding neurogenesis following neonatal injury. It is reported that the total number of new hippocampal SGZ-derived neurons is reduced bilaterally after neonatal ischemia [7]; however, another study reported that brain injury induced neurogenesis in neonatal animals, which was similar in adult animals, and H-I insult leads to a greater amount of neurogenesis in the DG than hypoxia alone [3]. The present study demonstrated increased cell proliferation in the DG following H-I. The observed difference in neurogenesis was likely due to the extent of injury in the model. Increased cell proliferation may play a role in the recovery of the neonatal animal from an H-I insult, and enhancement of proliferation may improve recovery of neurological deficits. bFGF is a pleotropic growth factor, and its levels are affected by multiple processes. It is a polypeptide with potent trophic and protective effects on the brain and has been reported to exert neuroprotection against a wide variety of insults, including ischemic neuronal injury [10,24]. Existing data indicate that bFGF can have both neuroprotective and neurorestorative properties [15]. Neuroinflammation downregulated protein expression of FGF2 and p-ERK1/2 in the hippocampus, and exogenous FGF2 alleviated impairment of hippocampal neurogenesis induced by neuroinflammation [16]. Our results revealed that increased cell proliferation in the DG of the hippocampus after H-I peaked at 7–14 days. Expression of bFGF mRNA is similar to that in BrdU-positive cells, indicating that proliferation in the hippocampus is correlated with levels of bFGF. An increase in FGF2 expression was observed in dorsal hippocampal astrocytes after acute stress. Astrocytesecreted FGF2 is necessary for stress hormone-induced enhancement in NPC proliferation in vitro [8]. The mechanisms of bFGF expression following neonatal H-I remain unexplored. It is reported that exogenous bFGF subcutaneously injected rapidly crosses the blood–brain barrier throughout life, regulating NPC proliferation during development as well as adulthood [22]. bFGF knockout mice exhibit a significant decrease in the number of newly generated neurons. However, the addition of bFGF to hippocampal slice cultures from knockout mice is unable to rescue the phenotype [23]. Our results revealed that exogenous bFGF slightly promotes cell proliferation in the hippocampal DG following neonatal H-I. Collectively, this indicated that bFGF is required for neurogenesis in the adult hippocampus and neonatal brain following damage but may function synergistically in combination with other intrinsic mechanisms or extrinsic growth factors. Furthermore, the time window may be important for pursuing future neurological therapies for clinical application following damage. The mechanism(s) of neuroprotection by bFGF, including direct cytoprotection as well as effects on the regional cerebral blood flow, remains unclear. One of the limitations of the present study was that we did not show the extent of HIBD in each group. However, we intend to demonstrate this in future studies.
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5. Conclusions Cell proliferation in the DG is induced by neonatal H-I, and bFGF promotes cell proliferation following neonatal H-I. It is likely that bFGF has neuroprotective and neurorestorative properties for neurological deficits following neonatal H-I. Declarations of interest None. Conflict of interest All authors declare that they have no conflict of interest.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Basic fibroblast growth factor enhances cell proliferation in the dentate gyrus of neonatal rats following hypoxic–ischemic brain damage ⁎
Huan Zhua,1, Lixing Qiaoa,1, Yao Sunb, Liping Yina, Li Huanga, Li Jianga, , Jiaqing Lic,
T
⁎
a
Department of Pediatrics, Zhongda Hospital, Southeast University, Nanjing, Jiangsu Province 210009, China Rehabilitation Department, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu Province 210008, China c Department of Anesthesiology, Children’s Hospital of Nanjing Medical University, Nanjing, Jiangsu Province 210008, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Cell proliferation Dentate gyrus Neonatal HIBD bFGF Cerebral hypoxia Cerebral ischemia
Background: Perinatal hypoxic–ischemic insult is considered a major contributor to child mortality and morbidity and leads to neurological deficits in newborn infants. There has been a lack of promising neurotherapeutic interventions for hypoxic–ischemic brain damage (HIBD) for clinical application in infants. The present study aimed to investigate the correlation between neurogenesis and basic fibroblast growth factor (bFGF) in the hippocampal dentate gyrus (DG) region in neonatal rats following HIBD. Material and methods: Cell proliferation was examined by detecting BrdU signals, and the role of bFGF in cell proliferation in the DG region following neonatal HIBD was investigated. Results: Cell proliferation was induced by HIBD in the hippocampal DG of neonatal rats. Furthermore, bFGF gene expression was upregulated in the hippocampus in neonatal rats, particularly between 7 and 14 days after HIBD. Moreover, intraperitoneal injection of exogenous bFGF enhanced cell proliferation in the hippocampal DG following neonatal HIBD. Conclusions: Taken together, these data indicate that cell proliferation in the DG could be induced by neonatal HIBD, and bFGF promotes proliferation following neonatal HIBD.
1. Introduction Perinatal hypoxic–ischemic injury leads to neurological deficits in newborn infants and is considered a major contributor to child mortality and morbidity [20]. Hypoxic–ischemic brain damage (HIBD) encompasses complex pathological and cellular injuries to the brain caused by hypoxia, ischemia, cytotoxicity, or a combination of these conditions [21]. Despite significant progress in elucidating the mechanism(s) underlying hypoxia–ischemia (H-I), there is currently a lack of promising neurotherapeutic interventions following HIBD for clinical application in infants. It is well-known that the brain has some capacity for neurogenesis in response to various types of damage that result in the loss of neurons in adult mammalian brains following H-I, particularly in two regions, the dentate gyrus (DG) subgranular zone (SGZ) and subventricular zone in the lateral ventricles [11]. Endogenous neurogenesis after ischemic injury produces new cells that can integrate into neural networks and play active roles in the recovery from neurological deficits [6]. However, there is contradictory evidence as to whether neonatal H-I increases or decreases the number of hippocampal precursor cells ⁎
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[3,7,25]. Endogenous neurogenic capacity depends on not only the intrinsic properties of neural precursor cells (NPCs) but also the regional differences in extrinsic regulatory signals and receptor expression. Therefore, harnessing the abilities of such extrinsic factors to amplify neurogenesis represents a neurotherapy for neonatal H-I. The basic fibroblast growth factor (bFGF, also called FGF2) is a member of the FGF superfamily proteins. It binds to the tyrosine kinase receptors FGFR1–4 to activate downstream signaling [24]. bFGF plays an important role in neurogenesis and also promotes neural stem cell (NSC) proliferation and differentiation into neurons during development as well as in the adult mouse brain via pharmacological addition or blockade of bFGF [4,12,22]. However, bFGF-deficient mice exhibit no obvious deficit in NSC proliferation in the adult DG but exhibit impairments in the differentiation of NPCs [23]. bFGF efficiently promotes the regeneration of neurons after injury [2,14] and has also been shown to modulate axonal branching and synaptic plasticity in vitro and in vivo [1,18]. As a neurotrophic factor, bFGF is highly significant for the development of therapies as it has the potential to restore neurological deficits in neurodegenerative diseases and acute stress [8,9,15]. However, the role
Corresponding authors. E-mail addresses: [email protected] (L. Jiang), [email protected] (J. Li). Co-first authors.
https://doi.org/10.1016/j.neulet.2018.01.046 Received 22 September 2017; Received in revised form 18 January 2018; Accepted 23 January 2018 Available online 20 March 2018 0304-3940/ © 2018 Elsevier B.V. All rights reserved.
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chosen for analysis. BrdU-positive cells were counted from the DG region in four 400 × magnification images in each section of the brain at the different time points.
of bFGF in neurogenesis following neonatal H-I remains unexplored. The present study aimed to investigate the correlation between neurogenesis and bFGF in the hippocampal DG region of neonatal rats following H-I. Our data indicated that cell proliferation in the DG is induced by neonatal H-I and that cell proliferation is promoted by bFGF following neonatal H-I. bFGF may have neuroprotective and neurorestorative properties for neurological deficits following neonatal H-I.
2.4. RT-PCR Isolation of total mRNA from the hippocampus was performed using TriPure reagent, following the one-step guanidinium isothiocyanate and acidified phenol method according to the manufacturer’s instructions. RNA concentration was determined using optical density measurements at 260 nm, and RNA purity and integrity were identified using an ultraviolet spectrometer and agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg of total RNA in a 20-μL reaction volume by reverse transcription insolution (4 μL MgCl2, 2 μL dNTP mixture, 1 μL OligodT-Adaptor primer, 0.5 μL RNase inhibitor, and 1 μL reverse transcriptase) at 42 °C for 30 min, 99 °C for 5 min, and 5 °C for 5 min. Ten microliters of product of the reverse transcription reaction were used as a template. PCR was run at 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 60 s, and finally at 72 °C for 8 min. Forward (5′-CTT TGG GTG GAA GGC TGG TCG-3′) and reverse (5′-TGC GGG AAG CGA AGT GAT GC-3′) primers were used to amplify a 448 bp rat bFGF fragment, and 308 bp GADPH served as a control with the following primers: sense 5′- TCA CTC AAG ATT GTC AGC AA-3′ and antisense 5′- AGA TCC ACG ACG GAC ACA TT-3′. Equal amounts of corresponding bFGF and GADPH products were loaded onto 1.5% agarose gels, and the optical densities of ethidium bromide-stained DNA bands were used to quantify the expression of the bFGF gene.
2. Material and methods 2.1. Materials Postnatal day 7 Sprague–Dawley (SD) rats (weighing 12.70 ± 1.14 g; male and female) were purchased from the Animal Laboratory at the Medical College of Southeast University (Nanjing, China). BrdU In-Situ Detection Kits were purchased from BD Bioscience. Oligonucleotide primers for bFGF and GADPH used for RT-PCR were synthesized by SANGON Co., Ltd. TriPure isolation reagent was purchased from Roche Diagnostics Corp. RNA PCR Kits (AMV) version 3.0 were purchased from TaKaRa Biotechnology Co., Ltd (Dalian). 2.2. Animals Procedures for the use of laboratory animals were approved by the institutional animal use and care committee, and the methods were conducted in accordance with the approved guidelines. For immunohistochemistry (IHC), SD rats were randomly divided into six groups (eight rats per group): sham, hypoxia, ischemia, H-I, normal saline (NS), and bFGF groups. Exogenous bFGF (10 μg/kg) was intraperitoneally injected every day for 7 days following H-I in the bFGF group, and saline was used as a control in the NS group. The rats were intraperitoneally injected with 100 mg/kg BrdU twice every week 48 h after different injuries. For RT-PCR, SD rats were randomly divided into four groups: sham, hypoxia, ischemia, and H-I groups. Animals were not injected with BrdU for RT-PCR analysis. H-I was induced according to previously described methods [13]. Seven-day-old SD rats were anesthetized by inhaling diethyl ether. The left common carotid artery was isolated and ligated using 4-0 type silk. The rats were placed in a hypoxic chamber (8% oxygen, 92% nitrogen) for 2 h, then in enclosed, vented chambers that were partially submerged in water (37.0 °C). Animals in the sham group were subjected to isolation but no ligation and no subsequent hypoxia, animals in the hypoxia group were only exposed to hypoxia and not ligation, and animals in the ischemia group were ligated but not exposed to hypoxia.
2.5. Statistical analysis Data are presented as mean ± standard deviation. Statistical comparisons were made using analysis of variance. The Student–Newman–Keuls q-test was used for comparisons between two groups. P < .05 was considered statistically significant. 3. Results 3.1. Pathological changes and apoptosis of brain cells following H-I The H-I model was produced according to the traditional Rice model [13]. Carotid artery ligation caused the ipsilateral eye to remain unopened after 90 days following H-I (Fig. 1a). Brains were collected, and atrophy of the left brain was observed (Fig. 1b). Brain damage of experimental animals in each group was compared at different time points, and hippocampal histopathological changes in each group were detected by the HE staining method. No evident damage was found in the control and sham operated groups (Fig. 1c). However, HE staining revealed edema and apoptosis of brain cells in the hippocampus, and the number of neurons was decreased at 24 h following H-I (Fig. 1d–f). The volume of the hippocampus was significantly reduced, and the pyramidal cells were randomly arranged. Necrosis was also observed after 5 days in the H-I model (Fig. 1g and h). The above data indicate neuronal damage in the neonatal brain following H-I.
2.3. Immunohistochemistry and hematoxylin and eosin (HE) staining The rats were sacrificed at 3, 7, 14, 21 days after injury for IHC and at 0 h, 6 h, 24 h, 48 h, 5 days, 7 days, and 90 days (n = 8) after injury for HE staining. Brains were removed, postfixed in 4% paraformaldehyde, and then cut into sections. Paraffin-embedded tissue was cut to 4μm-thick sections. For HE staining, sections were deparaffinized using xylene, rehydrated, stained using HE, dehydrated with alcohol, and cleared in xylene. Paraffin was removed from the sections using xylene according to the instructions on the BrdU In-Situ Detection Kit. Endogenous peroxidase activity was blocked in the sections. Antigen retrieval was performed using a microwave method. Biotinylated antiBrdU antibodies were applied to the sections on the slide and incubated for 1 h at 37 °C. Ready-to-use streptavidin–HRP was then applied to each slide and incubated for 30 min at room temperature. DAB substrates were stained in hematoxylin. Sections were dehydrated, cleared, and mounted. Results were considered positive when the cell nuclei were stained dark brown. Phosphate-buffered saline was used as a negative control in the procedure. The small intestines of BALB/c mice were stained as a positive control. For cell number quantitation, sections at 80 μm-intervals were
3.2. Time course of cell proliferation in the hippocampus of neonatal brains following H-I Endogenous neurogenesis was induced after ischemic injury in the adult mice brain. In order to investigate whether neurogenesis was also stimulated in neonatal brains following H-I, animals were intraperitoneally injected with BrdU and then sacrificed at specific time points after H-I. The number of newly generated cells was estimated in the entire DG region of the hippocampus by detecting the BrdU signal. The majority of BrdU-positive cells in the DG showed a dark brown color in the nuclei (Fig. 2a–d). There was no pronounced change in the 68
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Fig. 1. Pathological changes and apoptosis of brain cells following hypoxia–ischemia (H-I). (a) Carotid artery ligation caused the ipsilateral eye to remain unopened after 90 days following H-I. (b) Atrophy of the left brain after 90 days following H-I. (c) No evident damage was observed in the hippocampal dentate gyrus in the control and the sham operated groups. (d–f) Hematoxylin and eosin staining reveals edema and apoptosis of brain cells in the hippocampus and a decrease in the number of neurons at 24 h following H-I. Arrows indicate apoptotic bodies. (g and h) Reduction of the hippocampal volume and random pyramidal cell arrangement after 5 days following the H-I model. Necrosis was also observed. Arrows indicate apoptotic bodies. Scale bar represents 10 μm.
markedly reduced 14 days after H-I.
number of BrdU-labeled cells in the hippocampal DG of rats in the sham operated group at different time points (Fig. 2e). The number of BrdUpositive cells was significantly increased in the hippocampal DG of the hypoxia and ischemia groups at 7 or 14 days following damage (182.7% and 146.2% at 7 and 14 days, respectively, after hypoxia vs 3 days, P < .001; 204.5% and 161.6% at 7 and 14 days, respectively, after ischemia vs 3 days, P < .001), and H-I induced a greater number of BrdU-positive cells at different time points (184.9% vs sham group at 3 days, P < .001; 287.2% vs sham group at 7 days, P < .001; 236.4% vs sham group at 14 days, P < .001; 138.6% vs sham group at 21 days, P < .01) (Fig. 2e and Table 1). There was also a significantly higher density of BrdU incorporation between 7 and 14 days following H-I (217% and 195.4% at 7 and 14 days, respectively, after H-I vs at 3 days, P < .001) (Fig. 2 and Table 1). Taken together, these data indicate that cell proliferation was induced by H-I in the hippocampal DG of neonatal rats. Cell proliferation shows increased dynamics after 7 days and then
3.3. Increased expression of bFGF mRNA in the DG following H-I As a potent stem cell mitogen, bFGF promotes neurogenesis during development and in adult mice after injury. Acute stress increases astrocytic bFGF expression to mediate hippocampal cell proliferation [8]. We examined the expression of bFGF mRNA levels in the hippocampus following damage. There was no pronounced change in the bFGF mRNA levels in the hippocampus of rats in the sham operated group at different time points (Fig. 3a and e). bFGF mRNA levels were markedly increased in the hippocampus of the hypoxia and ischemia groups after 7 or 14 days following damage (169.6% and 134.2% at 7 and 14 d, respectively, after hypoxia vs 3d, P < .001 and P < .01; 208.1% and 141.3% at 7 and 14 days, respectively, after ischemia vs 3 days, P < .001 or P < .01), and H-I induced higher bFGF mRNA levels at 69
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Fig. 2. Time course of cell proliferation in the hippocampus of the neonatal brain following hypoxia–ischemia (H-I). (a–d) BrdU-positive cells in the hippocampal dentate gyrus (DG) at 3 days (a), 7 days (b), 14 days (c), and 21 days (d) following H-I. (e) Quantification of the number of BrdU-positive cells in the hippocampal DG at different time points following damage. H-I induced a greater number of BrdU-positive cells at different time points. The highest density of BrdU incorporation was detected between 7 and 14 days following H-I. **P < .01 ***P < .001 vs sham operated group, ## P < .01 ### P < .001 vs 3 days or 21 days, n = 8. Scale bar represents 10 μm. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
Table 1 The number of BrdU-positive cells in the left DG at different time points following damage (x ± S, N = 8). group
The number of BrdU-positive cell 3d
sham hypoxic ischemic hypoxic-ischemic
13.25 13.00 14.00 24.50
7d ± ± ± ±
3.84 3.74 4.50 5.07***
18.50 23.75 28.63 53.13
14 d ± ± ± ±
3.59 4.17***,### 4.89***,### 5.44***,###
20.25 19.00 22.63 47.88
21 d ± ± ± ±
4.40 3.93### 3.54### 5.25***,###
14.25 14.25 12.88 19.75
± ± ± ±
3.81 3.92 4.36 4.53**
** P < .01. *** P < .001 vs sham. ### P < .001 vs 3d or 21d.
neurogenesis following neonatal H-I, rats were intraperitoneally injected with bFGF for 7 days following H-I, and saline was injected in the control group. Brains were removed, and BrdU-labeled cells were detected in the hippocampus. Exogenous bFGF slightly increased the number of BrdU-labeled cells in the hippocampus at 7 and 14 days following H-I compared with those in the H-I and NS groups (119.8% vs H-I group, 121.9% vs NS group at 7 days, P < .05) (Fig. 4). This suggested that exogenous bFGF promotes cell proliferation in the hippocampal DG following neonatal H-I. Taken together, these data indicated that cell proliferation in the DG may be induced by neonatal H-I and that bFGF promotes proliferation following neonatal H-I.
different time points (149.4% vs sham group at 3 days, P < .001; 302.0% vs sham group at 7 days, P < .001; 256.6% vs sham group at 14 days, P < .001; 137.3% vs sham group at 21 days, P < .01) (Fig. 3b–e). Furthermore, the highest levels of bFGF mRNA were detected between 7 and 14 days following H-I (208.9% and 198.9% at 7 and 14 days, respectively, after H-I vs at 3 days, P < .001) (Fig. 3d and e). These data indicated that bFGF gene expression is upregulated in the hippocampus following H-I in neonatal rats, particularly between 7 and 14 days after H-I, which is consistent with the time course of cell proliferation indicated by BrdU incorporation. 3.4. Exogenous bFGF promotes cell proliferation following neonatal H-I In order to investigate whether bFGF also plays a role in 70
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Fig. 3. Increase in the expression of basic fibroblast growth factor (bFGF) mRNA levels in the dentate gyrus following hypoxia–ischemia (H-I). (a) No evident change in bFGF mRNA levels in the hippocampus of rats in the sham operated group at different time points. (b–d) Increase in the expression of bFGF mRNA levels in the hippocampus of hypoxia, ischemia, and H-I groups after 7 or 14 days following damage. (e) Quantification of bFGF mRNA levels at different time points in the hippocampus following damage. H-I induced a higher level of bFGF mRNA at different time points. The highest level of bFGF mRNA was detected between 7 and 14 days following H-I. GAPDH was used as a loading control. **P < .01 ***P < .001 vs sham operated group, ## P < .01### P < .001 vs 3 days or 21 days, n = 4.
4. Discussion
Therefore, it is usually used for birth dating and identification of proliferating cells. BrdU incorporation into DNA produces toxic and mutagenic side effects. The integration of a bromine atom into the DNA alters its stability, increasing the risk of sister-chromatid exchanges, mutations, DNA double-strand breaks and also lengthening the cell cycle of the cells that incorporate it. BrdU staining can be performed by immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BrdU [17]. BrdU immunohistochemistry is the most popular technique to confirm neurogenesis in the adult mammalian brain, including in humans; therefore, we used this technique to provide evidence for an increase in cell proliferation in the neonatal hippocampus following H-I. Most research has focused on the hippocampus to study neurogenesis during development and in the adult brain following injury and further investigate the susceptibility to damage and regenerative capacity due to NSCs in the hippocampus [3,7,11,25]. In the mammalian brain, proliferation is maintained at a high level in the early postnatal brain, particularly in the hippocampus, cerebellum, and olfactory bulb.
H-I is a major cause of morbidity and mortality in the perinatal period, with an incidence of 1/4000 live births [19]. To date, there is sufficient evidence showing the etiology of perinatal asphyxial brain injury, and significant progress has been made to understand the mechanism(s) underlying H-I. However, promising neurotherapeutic interventions after damage must be developed for clinical application in infants. There is increasing evidence that indicates a significant increase in neurogenesis following certain types of damage, such as asphyctic, ischemic, traumatic, and epileptogenic brain injuries, indicating that instead of minimizing cell death, neurological deficits may be improved by stimulation of endogenous neurogenesis after injury [5]. In the present study, we observed that cell proliferation in the DG may be induced after neonatal H-I and that bFGF promoted this cell proliferation. BrdU is a thymidine analog that can incorporate into newly synthesized DNA of dividing cells during the S-phase of the cell cycle.
Fig. 4. Exogenous basic fibroblast growth factor (bFGF) promotes cell proliferation following neonatal hypoxia–ischemia (H-I). Quantification of the number of BrdU-positive cells in the hippocampal dentate gyrus of bFGF-injected mice at different time points following damage. Exogenous bFGF slightly increased the number of BrdU-labeled cells in the hippocampus at 7 d following H-I, compared with those in the H-I and normal saline (NS) groups. **P < .01 ***P < .001 vs sham operated group, ## P < .01 ### P < .001 vs 3 days or 21 days, and *P < .05 vs NS group, N = 8.
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Acknowledgement
The DG produces significant numbers of new neurons throughout life. There is inconsistent evidence regarding neurogenesis following neonatal injury. It is reported that the total number of new hippocampal SGZ-derived neurons is reduced bilaterally after neonatal ischemia [7]; however, another study reported that brain injury induced neurogenesis in neonatal animals, which was similar in adult animals, and H-I insult leads to a greater amount of neurogenesis in the DG than hypoxia alone [3]. The present study demonstrated increased cell proliferation in the DG following H-I. The observed difference in neurogenesis was likely due to the extent of injury in the model. Increased cell proliferation may play a role in the recovery of the neonatal animal from an H-I insult, and enhancement of proliferation may improve recovery of neurological deficits. bFGF is a pleotropic growth factor, and its levels are affected by multiple processes. It is a polypeptide with potent trophic and protective effects on the brain and has been reported to exert neuroprotection against a wide variety of insults, including ischemic neuronal injury [10,24]. Existing data indicate that bFGF can have both neuroprotective and neurorestorative properties [15]. Neuroinflammation downregulated protein expression of FGF2 and p-ERK1/2 in the hippocampus, and exogenous FGF2 alleviated impairment of hippocampal neurogenesis induced by neuroinflammation [16]. Our results revealed that increased cell proliferation in the DG of the hippocampus after H-I peaked at 7–14 days. Expression of bFGF mRNA is similar to that in BrdU-positive cells, indicating that proliferation in the hippocampus is correlated with levels of bFGF. An increase in FGF2 expression was observed in dorsal hippocampal astrocytes after acute stress. Astrocytesecreted FGF2 is necessary for stress hormone-induced enhancement in NPC proliferation in vitro [8]. The mechanisms of bFGF expression following neonatal H-I remain unexplored. It is reported that exogenous bFGF subcutaneously injected rapidly crosses the blood–brain barrier throughout life, regulating NPC proliferation during development as well as adulthood [22]. bFGF knockout mice exhibit a significant decrease in the number of newly generated neurons. However, the addition of bFGF to hippocampal slice cultures from knockout mice is unable to rescue the phenotype [23]. Our results revealed that exogenous bFGF slightly promotes cell proliferation in the hippocampal DG following neonatal H-I. Collectively, this indicated that bFGF is required for neurogenesis in the adult hippocampus and neonatal brain following damage but may function synergistically in combination with other intrinsic mechanisms or extrinsic growth factors. Furthermore, the time window may be important for pursuing future neurological therapies for clinical application following damage. The mechanism(s) of neuroprotection by bFGF, including direct cytoprotection as well as effects on the regional cerebral blood flow, remains unclear. One of the limitations of the present study was that we did not show the extent of HIBD in each group. However, we intend to demonstrate this in future studies.
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5. Conclusions Cell proliferation in the DG is induced by neonatal H-I, and bFGF promotes cell proliferation following neonatal H-I. It is likely that bFGF has neuroprotective and neurorestorative properties for neurological deficits following neonatal H-I. Declarations of interest None. Conflict of interest All authors declare that they have no conflict of interest.
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